We have developed a method for extracellular labeling of secreted growing peptide chains in bacteria, and have thereby unequivocally demonstrated the secretion of a growing chain across a membrane. We have also observed that several secreted proteins are synthesized by the membrane-polysome fraction and cytoplasmic proteins by the free polysome fraction. We plan to use this method to determine how generally proteins are secreted, and membrane proteins are inserted, while growing. In particular we shall test for extracellular labeling of proteins (toxins, colicins) that have to cross the outer membrane of gram-negative bacteria. The most challenging question -- the mechanism of transport of the chain across the membrane -- will be studied by the use of mutants altered in secretion, cells with altered lipid composition, a search for an energy requirement, and cross-linking of protruding growing chains or bound ribosomes to adjacent membrane proteins. The role of the leader sequence and a possible membrane receptor, in the binding of ribosomes (via their nascent chain) to membrane, will be studied by reconstituting membrane-polysome complexes in vitro from polysomes and various naturally occurring or modified membranes. Finally, we shall seek an explanation for the surprisingly large fraction of ribosomes (ca. 50%) with an apparently physiological attachment to membrane.